This invention relates to a novel synthetic substrate for assaying peptidase activity and an assay method of peptidase. More particularly, the present invention relates to an amide compound of the formula ##STR2## wherein R is L-leucyl or .gamma.-L-glutamyl and X and Y are the same or different, and are halogen atom or salt thereof, and an assay method of peptidase which comprises:
An assay method of enzyme activity which comprises treating an amide compound of the formula ##STR3## wherein R.sub.1 is L-leucyl or .gamma.-L-glutamyl group, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxyl, carboxyl or sulfo group and R.sub.5 and R.sub.6 in together may be constituted the carbon ring, or water soluble salt thereof with peptidase, treating the thus liberated amine of the formula ##STR4## wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 have the same meanings hereinabove, with oxidase which consumes oxygen and forms pigment in the presence of a coupler of the formula ##STR5## wherein R.sub.7 is hydrogen, amino, substituted amino or hydroxyl group, R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 are hydrogen, halogen, lower alkyl, lower alkoxy, amino, substituted amino, hydroxyl, carboxyl or sulfo group, and R.sub.11 and R.sub.12, in together, may optionally be carbon ring, or R.sub.7 may optionally be hydrogen when at least one of R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 is amino, substituted amino or hydroxyl, then quantitatively measuring the detectable changes, and an assay method which comprises treating an amide compound of the formula ##STR6## wherein R is L-leucyl or .gamma.-L-glutamyl group, and X and Y are the same or different and are halogen atom, or salt thereof, with peptidase in a sample to liberate aniline derivative of the formula ##STR7## wherein X and Y have the same meanings hereinbefore, oxidizing the said aniline derivative, and quantitatively measuring the detectable
Peptidase has been known as a general term for enzyme which acts on peptide bond in L-peptide and splitting at N-terminal to liberate amino acids or lower peptides. For example, aminopeptidase such as leucine aminopeptidase (True LAP in clinical chemistry) or arylamidase (Clinical LAP in clinical chemistry, hereinafter sometimes designates AA)(hereinafter True LAP and Clinical LAP are generically designates as LAP), cystine aminopeptidase, proline aminopeptidase, arginine aminopeptidase, alanine aminopeptidase or .gamma.-glutamyl transpeptidase (.gamma.-GTP) has been known.
Among the enzyme hereinabove, LAP and .gamma.-GTP are widely distributed in the tissues in vivo, and in serum. These enzymes are increased in disease condition and are important marker for clinical diagnosis and important items for enzyme activity assay in clinical determination.
LAP is an enzyme which hydrolyses amino-terminal residue of L-peptide having L-leucine or peptide related thereto as N-terminal residue to liberate L-leucine, and is distributed in tissue in vivo and in serum. An amount of LAP in serum is varied depending upon the conditions of body, and is increased at acute hepatitis, hepatoma, metastatic hepatoma, hepatocirrhosis and cholangitis. Therefore, LAP activity is a marker of these symptoms, and assay of LAP activity is indispensable for clinical diagnosis of these symptoms.
.gamma.-GTP is an enzyme relating to metabolism of .gamma.-glutamylpeptide in vivo and catalyzes the reaction of hydrolyzing the .gamma.-glutamyl group in .gamma.-glutamyl peptides and transferring the said group to other amino acids or peptides, and is widely distributed in tissues in vivo and in serum. .gamma.-GTP in serum varies depending on the conditions of symptoms. The clinical value of serum .gamma.-GTP is said to be higher in cholestatic hepatitis, obstructive jaundice and primary metastatic hepatoma and active chronic hepatitis, and lower in non-active chronic hepatitis, The determination of serum .gamma.-GTP activity is specific for chronic hepatitis and is useful for clinical diagnosis and pathological ascertainment of these diseases.
Heretofore assay method of LAP has been known, most of those are colorimetric assay method of liberated amine compound for LAP activity. In the assay, L-leucyl-p-nitroanilide is used as synthetic substrate and yellow color of p-nitroaniline generated by enzymatic action of LAP is measured, however at the colorimetric determination of the said color, color absorption maximum is disadvantageously overlapped, moreover serum component such as bilirubin pigment affects disadvantageously. Also, a method using synthetic substrate L-leucyl-.beta.-naphthylamide has been known, however the said method has number of disadvantages. The assay method is quite complex and required precise performance, for example the formed .beta.-naphthylamine is coupled with 5-nitro-2-aminomethoxybenzene diazotate to form pigment, or .beta.-naphthylamine thus formed is diazotated with sodium nitrite to couple with N-(1-naphthyl)-ethylenediamine or is condensated with p-dimethylaminobenzaldehyde or p-dimethylamino cinnamaldehyde to form pigment which is colorimetrically assayed. Further, the standard substance, .beta.-naphthylamine, is toxic for inducing carcinoma or tumor of bladder.
Assay methods of .gamma.-GTP are known and most of them are colorimetric assay of a liberated amine compound from synthetic substrate by .gamma.-GTP. For example an assay method using .gamma.-glutamyl-p-nitroanilide is that .gamma.-glutamyl-p-nitroanilide is treated with .gamma.-GTP to liberate yellow-colored p-nitroaniline which is colorimetrically measured at 410 nm. The absorbancy at 410 nm is inhibited by compound in body fluids, especially bilirubin. In order to avoid the effect of bilirubin pigment, control assay for specimen should be strictly and exactly measured, which causes disadvantages. Also liberated p-nitroaniline is condensed with an aldehyde such as p-dimethylamino cinnamaldehyde or p-dimethylaminobenzaldehyde to produce a color which is colorimetrically assayed at long-wave length red color. This colorimetric assay procedure is affected by temperature for coloring sensitivity and so is troubles for reproducibility. Further, a method wherein the produced p-nitroaniline is diazotized and condensed with 3,5xylenol and the thus formed red color is measured, is provided. This assay method, however, is quite complex and required multi reaction steps. Furthermore, an assay method using synthetic substrate .gamma.-L-glutamyl-.beta.-naphthylamide is provided. In this method, liberated .beta.-naphthylamine by .gamma.-GTP is changed to the diazonium salt and is colorimetrically measured or is reacted with 3-methyl-2-benzothiazolinone hydrazone and oxidizing agent to produce color which is colorimetrically measured, or the aldehyde compound of coloring agent hereinbefore is condensed therewith to assay colorimetrically. In these methods, carcinogenicity of .beta.-naphthylamine causes problem, and also these assay method requires strict control of complex reaction process which causes disadvantages.
We had previously found that amide compound of the formula (which does not exist in serum) ##STR8## wherein R' is hydroxyl, amino or di-lower alkylamino and R" is hydrogen, lower alkyl or carboxyl, is excellent synthetic substrate for LAP and liberates amine compound by the action of LAP. The said amine compound is oxidized by the action of oxidase such as ascorbate oxidase or laccase and oxygen is consumed to form chromogen. LAP activity can be measured by measuring the amount of consumed oxygen or produced chromogen.
The assay method using the above synthetic substrate has disadvantages in which absorption maximum of chromogen is no more than 550 nm and is affected the contaminant in body fluid, and so is required the substrate which forms chromogen of higher absorption maximum.